Download MagJET Plant RNA Kit - Thermo Fisher Scientific

PRODUCT INFORMATION
Thermo Scientific
MagJET Plant RNA Kit
#K2771, #K2772
Read Storage information (p. 4) upon receipt and store kit
components appropriately!
www.thermoscientific.com/onebio
#K2771
Lot 00000000
Exp. 00.0000
CERTIFICATE OF ANALYSIS
Thermo Scientific™ MagJET™ Plant RNA Kit is qualified by isolation of total RNA from
50 mg of plant tissue following the protocol outlined in the manual. The quality of isolated RNA
is evaluated spectrophotometrically and by agarose gel electrophoresis. The purified RNA has
an A260/A280 ratio of 2.0±0.2 and the RNA integrity number (RIN) of ≥7.
Quality authorized by:
Rev.1
Jurgita Žilinskienė
f
2
CONTENTS
page
COMPONENTS OF THE KIT ....................................................................................................4
STORAGE .................................................................................................................................4
DESCRIPTION ..........................................................................................................................4
PRINCIPLE ............................................................................................................................... 4
IMPORTANT NOTES ................................................................................................................5
ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED......................................................6
AVOIDING RIBONUCLEASE CONTAMINATION .....................................................................6
RNA HANDLING AND STORAGE ............................................................................................ 6
STARTING MATERIAL HANDLING AND SAMPLE HOMOGENIZATION.................................7
PROTOCOL SELECTION GUIDE ............................................................................................. 9
TOTAL RNA PURIFICATION PROTOCOLS AND PIPETTING INSTRUCTIONS ................... 10
Protocol A. Instructions for total RNA purification from up to 50 mg plant sample
using KingFisher Flex 96 and Microtiter deep well 96 plates ............................................... 10
Protocol B. Instructions for total RNA purification from up to 50 mg plant sample
using KingFisher Duo with 12-pin magnet head and Microtiter deep well 96 plate .............. 12
Protocol C. Instructions for manual plant total RNA purification from up to 50 mg
plant sample........................................................................................................................ 14
TROUBLESHOOTING ............................................................................................................ 15
SAFETY INFORMATION ........................................................................................................ 16
3
COMPONENTS OF THE KIT
MagJET Plant RNA Kit
Lysis Buffer for MagJET Plant RNA Kit
MagJET Magnetic Beads
DNase I (lyophilized)
DNase I Reconstitution Buffer
2X DNase I Buffer
Manganese Chloride Solution
Wash Buffer 1 (conc.) for MagJET Plant RNA Kit
Wash Buffer 2 (conc.) for MagJET Plant RNA Kit
Rebinding Buffer for MagJET Plant RNA Kit
Water, nuclease free
#K2771
96 preps
72 mL
2 × 1.4 mL
1 vial
1 mL
12 mL
3 × 1 mL
110 mL
50 mL
20 mL
30 mL
#K2772
384 preps
2 × 140 mL
10.6 mL
4 vials
2 × 1 mL
45 mL
9 × 1 mL
3 × 110 mL
3 × 50mL
70 mL
125 mL
STORAGE
When the kit is delivered, remove DNase I (lyophilized), DNase I Reconstitution Buffer and
Manganese Chloride Solution and store at -20°C. Reconstituted DNase I should be stored
at -20°C. MagJET Magnetic Beads should be stored at 4C. Other components of the kit
should be stored at room temperature (15-25°C).
DESCRIPTION
The MagJET RNA Kit is designed for automated high throughput or manual purification of total
RNA from a wide variety of plant species and tissue types.
The kit utilizes paramagnetic bead technology enabling high yields and robust performance.
High binding capacity, uniform particle size, and rapid magnetic response of MagJET magnetic
beads makes the technology ideal for high throughput automatic nucleic acid purification, as
well as for manual nucleic acid purification by low sample throughput users.
The resulting high quality purified RNA is free of proteins, nucleases and other contaminants or
inhibitors, and can be used in a wide range of downstream applications, such as RT-PCR,
RT-qPCR and other enzymatic reactions. See Table 1 for typical total RNA yields from various
sources.
PRINCIPLE
The MagJET Plant RNA Kit uses the highly efficient MagJET magnetic particle-based
technology for nucleic acid purification. The nucleic acid isolation process combines the simple
steps of sample lysis, RNA binding to the magnetic beads, DNA digestion, washing and
elution.
Purification protocols optimized for automated KingFisher instruments utilize a high throughput
magnetic bead transfer technique, where magnetic beads are transferred through different
reagent plates containing lysis, binding, washing and elution reagents. This enables high
throughput nucleic acid purification and eliminates multiple pipetting steps.
Alternatively, a protocol is provided where buffers and other reagents are transferred in each of
the protocol steps, while magnetic beads remain captured on the wall of a tube using a
magnetic rack. This allows the kit to be used in various throughput applications using a
magnetic rack and manual or automated pipetting equipment.
4
Table 1. Typical yields of total RNA from various sources.
Plant
Tissue (50 mg)
RNA yield, µg
leaf
leaf
leaf
leaf
leaf
leaf
leaf
leaf
leaf
seeds
leaf
stalk
seeds
leaf
seeds
fruit
stalk
stalk
seeds
18-20
15-20
21-52
25-32
25-36
24-30
8-12
16-19
13-20
20-40
11-19
22-28
30
30-40
44
18
10
4-5
40
Arabidopsis thaliana
Nicotiana tabacum
Tomato
Barley seedlings
Wheat shoots
Spinach
Onion
Rice
Corn
Canola (Rape)
Soy
Cucumber
Potato
Sunflower
IMPORTANT NOTES
 Add the indicated volumes of ethanol (96-100%) to Wash Buffer 1 (conc.) and Wash
Buffer 2 (conc.) prior to first use:
Concentrated buffer
Ethanol (96-100%)
Total volume:
#K2771 and #K2772
Wash Buffer 1
Wash Buffer 2
110 mL
50 mL
110 mL
200 mL
220 mL
250 mL
After preparing each solution, mark the bottle to indicate that this step has been completed.
 To prepare the DNase I solution add 0.44 mL of DNase I Reconstitution Buffer to each
vial of DNase I (lyophilized) and incubate at room temperature for 5 minutes. Occasional
gentle rotation of the vial helps to dissolve the DNase I, but avoid forceful mixing. Do not
vortex! Store at -20°C.
 Before each RNA purification, prepare a fresh aliquot of Lysis Buffer supplemented with
2 M DTT solution. Add 20 µL of 2 M DTT to each 1 mL of Lysis Buffer needed.
 Check all solutions in the kit for salt precipitation before each use. Re-dissolve any
precipitates by warming the solution at 37 °C, and then equilibrate to room temperature
(20 ± 5°C).
 Wear gloves when handling the Lysis Buffer, Rebinding Buffer and Wash Buffer 1 as
these reagents contain irritants (see page 16 for SAFETY INFORMATION).
5
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ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED
2 M DTT (dithiotreitol) solution (store at -20°C).
polyvinylpyrrolidone (PVP)(for RNA purification from woody, lignified and or polyphenol-rich
samples, see p.7-8).
Sodium chloride (NaCl) (for RNA purification from soy beans, see p.7-8).
96-100% ethanol, molecular biology grade.
Pipettes and RNase-free pipette tips.
8 or 12 channel pipettes and pipette tips.
RNase-free 1.5-2 mL plastic tubes.
Disposable gloves.
Thermomixer.
Centrifuge capable of ≥ 16,000 × g for microtubes.
Centrifuge capable of 3,000-4,000 × g with swinging-bucket rotor for 96-well plates.
Incubator or water bath capable of 56°C.
Vortex.
Mortar and pestle (for manual tissue disruption in liquid nitrogen).
Liquid nitrogen.
Commercial homogenizer and stainless steel beads (for mechanical automatic plant tissue
disruption).
Automatic magnetic particle processor and consumables.
For manual protocol acquire magnetic particle processing rack.
AVOIDING RIBONUCLEASE CONTAMINATION
RNA purity and integrity is essential for downstream applications. RNA can be degraded by
RNase A, which is a highly stable contaminant found in any laboratory environment. Care must
be taken not to introduce RNases into RNA preparations, especially during wash with Wash
Buffer 2 and elution steps. General recommendations to avoid RNase contamination include
the following:
 Wear gloves when handling reagents and RNA samples, as skin is a common source of
RNases. Change gloves frequently.
 Use sterile, disposable RNase-free pipette tips.
 Use appropriate reagents to remove RNase contamination from nondisposable items
(pipettes, centrifuges) and work surfaces.
 Keep all kit components tightly sealed when not in use. After usage close bottles
immediately.
RNA HANDLING AND STORAGE
 Keep the RNA on ice after extraction and while working with it.
 Store the extracted RNA at -20°C or -70°C. For long term use store RNA at -70°C.
6
STARTING MATERIAL HANDLING AND SAMPLE HOMOGENIZATION
 To minimize RNA degradation, avoid repeated freezing and thawing of the samples and
perform extractions from fresh material, or material that has been immediately frozen and
stored at -70°C.
 Appropriate sample storage is essential for reproducibility and high RNA yields. Yields of
RNA may vary depending on sample age, type of sample, and storage conditions.
 Efficient homogenization of the sample material is an essential step before RNA purification
in order to gain a good yield of high-quality RNA. The lysis procedure of plant tissue is most
effective with well homogenized, powdered samples. Plant tissue can be homogenized
using a mortar and pestle in the presence of liquid nitrogen or using commercial
homogenizers with steel beads.
Manual plant tissue homogenization
1. Place the plant sample in a mortar with liquid nitrogen. Grind the tissue using a pestle.
2. Allow the liquid nitrogen to evaporate and transfer the powder sample (10- 50 mg) into a
1.5 mL microcentrifuge tube containing 600 µL Lysis Buffer supplemented with DTT as
described on page 5.
 Transfer the homogenized tissue to the Lysis Buffer as quickly as possible to avoid
RNA degradation.
 All homogenized material must be thoroughly mixed with the Lysis Buffer. RNA
degradation can occur in particles that are left to dry on the walls of the tube.
3. Vortex for 10-20 s to mix thoroughly.
4. Incubate for 5 min at 56°C.
5. Centrifuge for 10 min at maximum speed (≥16,000 × g).
6. Carefully take 400 µL of the cleared supernatant and immediately proceed with the RNA
isolation according to protocols A - C.
Notes for lysis of different plant samples:
 For seed samples, use 10-20 mg starting material into the Lysis Buffer.
 To purify total RNA from woody, lignified and or polyphenol-rich samples such as branches,
twigs, needles, wax-coated leaves (such as laurel), supplement the Lysis Buffer with
polyvinylpyrrolidone (PVP) at a 2% (w/v) final concentration.
 To purify total RNA from soy beans, supplement the Lysis Buffer with sodium chloride
(NaCl) at a 2 M final concentration.
7
Mechanical (automatic) tissue disruption
Plant tissue can be homogenized with commercial homogenizers and grinding mills (with
stainless steel beads). High-throughput homogenizers offer a suitable method for handling up
to 96 samples simultaneously.
1. Place up to 50 mg of plant sample (or up to 20 mg for seeds) into microtube containing a
stainless steel bead and 600 µL Lysis Buffer supplemented with DTT as described on
page 5.
2. Grind the sample according to homogenizer instructions.
3. Incubate for 5 min at 56°C.
4. Centrifuge for 10 min at maximum speed (≥16,000 × g for microtube or 3,000–4,000 × g
for racked 96-well collection microtubes).
5. Carefully take 400 µL of the cleared supernatant and immediately proceed with the RNA
isolation according to protocols A – C.
Notes for lysis of different plant samples:
 Use 10-50 mg of fresh plant sample for mechanical automatic disruption (with stainless
steel beads) in single microtubes*.
 Use 10-20 mg plant lsample for mechanical automatic disruption (with stainless steel
beads) in racked 96-well collection microtubes*.
 Use 10-20 mg of plant seeds as starting material for mechanical automatic disruption (with
stainless steel beads) in racked 96-well collection microtubes and/or single mictrotubes*.
 To purify total RNA from woody, lignified and or polyphenol-rich samples such as branches,
twigs, needles, wax-coated leaves (e.g. laurel), supplement the Lysis Buffer with
polyvinylpyrrolidone (PVP) at a 2% (w/v) final concentration.
 To purify total RNA from soy beans, supplement the Lysis Buffer with sodium chloride
(NaCl) at a 2 M final concentration.
*- Proceed according homogenizer manufacturer recommendations.
8
PROTOCOL SELECTION GUIDE
The MagJET Plant RNA Kit provides optimized protocols for total RNA purification from
different amounts of starting material (10-50 mg). The Kit is compatible with automated and
manual sample processing, allowing high and low-throughput nucleic acid purification
workflows.
The following selection guide summarizes the available protocols depending on starting
sample weight, throughput and sample processing method. Automation protocols are
optimized for KingFisher Flex and KingFisher Duo instruments.
10-50 mg
(10-20 mg
of seeds)
Manual
processing
10-50 mg
(10-20 mg
of seeds)
KingFisher Duo
Instrument
Plant
homogenized
with liquid
nitrogen (see
the detailed
instruction on
page 7)
Plant
homogenized
with equipment
for disrupting
plant tissue*
(see the detailed
instruction on
page 8)
Sample
weight
KingFisher Flex
Instrument
Plant
homogenization
type
Throughput
per run
Protocol selection guide:
MagJET
purification
protocol
Up to 96
●
-
-
Protocol A
10
Up to 12
-
●
-
Protocol B
12
variable
-
-
●
Protocol C
14
Up to 96
●
-
-
Protocol A
10
Up to 12
-
●
-
Protocol B
12
variable
-
-
●
Protocol C
14
*Sample quantity may depend on the homogenizer type and throughput. Please proceed according
homogenizer manufacturer recommendations.
9
Page
TOTAL RNA PURIFICATION PROTOCOLS AND PIPETTING INSTRUCTIONS
Protocol A. Instructions for total RNA purification from up to 50 mg plant sample using
KingFisher Flex 96 and Microtiter deep well 96 plates
Note:
 When using the MagJET Plant RNA Kit for the first time, prepare working solutions of Wash
Buffer 1 and Wash Buffer 2 and reconstitute DNase I as described on page 5.
 Transfer the Plant_RNA_Flex protocol file to the KingFisher instrument before first use.
The instructions for transferring the protocol can be found in Chapter 4: “Using the software”
in the BindIt Software for KingFisher Instruments version 3.2 User Manual. The protocol
files for MagJET Plant RNA Kit can be found on the product web page on
www.thermoscientific.com/onebio
1. Homogenize the plant samples and prepare lysates as described on page 7-8 (SAMPLE
HOMOGENIZATION).
2. Prepare DNase l master mix for the number of samples to be processed.
DNase l master mix for purification of one sample (200 µL): 100 µL 2X DNase l Buffer,
4 µL DNase I (reconstituted), 20 µL Manganese Chloride Solution and 76 µL nuclease
free water. Aliquot prepared master mix to the DNase l plate (plate number 3, below).
3. Obtain six empty Thermo Scientific Microtiter deep well 96 plates and two empty Thermo
Scientific KingFisher Flex 96 KF plates.
4. Fill the plates as follows:
Plate
number
Plate type
Plate name
Content
Volume per
well
Wash Buffer 1
(supplemented with ethanol)
700 µL
DNase l master mix
200 µL
2
Microtiter deep well 96 plate Wash 1_1
3*
Microtiter deep well 96 plate
4
Microtiter deep well 96 plate Wash 1_2
Wash Buffer 1
(supplemented with ethanol)
700 µL
5
Microtiter deep well 96 plate Wash 2_3
Wash Buffer 2
(supplemented with ethanol)
700 µL
6
Microtiter deep well 96 plate Wash 2_4
Wash Buffer 2
(supplemented with ethanol)
700 µL
7
KingFisher Flex 96 KF plate
Elution
Water, nuclease free
100 µL
8
KingFisher Flex 96 KF plate
Tip plate
-
-
DNase l
*For best results, avoid storage of DNase I in 1X Reaction Buffer with MgCl2 at room temperature for
extended periods of time. It is recommended to prepare the DNase I plate just prior to use.
10
5. Prepare the Sample plate (Microtiter deep well 96 plate).
Add the following reagents to the Sample plate.
Plate
number
1
Plate type
Microtiter deep well 96 plate
Plate name
Sample
Content
Volume per
well
Plant tissue lysate
400 µL
Magnetic Beads*
25 µL
Ethanol
(96-100%)
400 µL
*-Resuspend Magnetic Beads well by vortexing before use.
6. Place a Thermo Scientific KingFisher Flex 96 tip comb for deep well magnets on a
Tip Plate (an empty KingFisher Flex 96 KF plate).
7. Start the Plant_RNA_Flex protocol with the KingFisher Flex 96 and load the plates
according to the KingFisher display. After all the plates have been loaded into the
instrument, the protocol will begin.
8. When the KingFisher Flex pauses at the dispense step (after the DNase l digestion,
approximately 30 minutes after starting the run), remove the DNase l plate from the
instrument and add 150 µL of Rebinding Buffer (concentrated) and 400 µL of ethanol
(96-100%) per well to rebind the RNA.
Plate
number
Plate type
Plate name
3
Microtiter deep well 96 plate
DNase l
Volume per
well
Rebinding Buffer (conc.)
150 µL
Ethanol
400 µL
(96-100%)
Content
9. Place the DNase l plate back into the instrument and press Start. After the pause, the
protocol will continue through to completion.
10. After the protocol is complete, remove the plates according to the instructions on the
KingFisher Flex display and turn off the instrument. Transfer the purified RNA from the
Elution plate to a fresh RNase-free microtiter plate. Keep on ice for immediate use in
downstream applications or store at -70°C.
11
Protocol B. Instructions for total RNA purification from up to 50 mg plant sample using
KingFisher Duo with 12-pin magnet head and Microtiter deep well 96 plate
Note:
 When using the MagJET Plant RNA Kit for the first time, prepare working solutions of Wash
Buffer 1 and Wash Buffer 2 and reconstitute DNase I as described on page 5.
 Ensure you are using the KingFisher Duo 12-pin magnet head and heating block
 Transfer the Plant_RNA_Duo protocol file to the KingFisher instrument before first use. The
instructions for transferring the protocol can be found in Chapter 4: “Using the software” in
the BindIt Software for KingFisher Instruments version 3.2 User Manual. The protocol files
for MagJET Plant RNA Kit can be found on product web page on
www.thermoscientific.com/onebio
1. Homogenize the plant samples and prepare lysates as described on page 7-8 (SAMPLE
HOMOGENIZATION).
2. Prepare DNase l master mix for the number of samples to be processed.
DNase l master mix for purification of one sample (200 µL): 100 µL 2X DNase l Buffer,
4 µL DNase I (reconstituted), 20 µL Manganese Chloride Solution and 76 µL nuclease
free water. Aliquot prepared master mix to the Microtiter deep well 96 plate row A (DNase l
row).
3. Obtain one empty Thermo Scientific Microtiter deep well 96 plate and one Thermo Scientific
KingFisher Duo Elution strip.
4. Prepare the Plant RNA plate (Microtiter deep well 96 plate).
Add the following reagents to the rows. Note that row B is reserved for the tips and should be
left empty. Note that row H is left empty.
Plate name and type
Plant RNA plate
Microtiter deep well
96 plate
Row
Row name
Content
A
B
DNase l
Tip
C*
Sample
DNase l master mix
12-tip comb
Plant tissue lysate
Magnetic Beads
Ethanol
(96-100%)
Wash Buffer 1
Wash Buffer 1
Wash Buffer 2
Wash Buffer 2
Empty
D
E
F
G
H
Wash 1_1
Wash 1_2
Wash 2_3
Wash 2_4
Empty
Sample/reagent
volume per well
200 µL
N/A
400 µL
25 µL
400 µL
700 µL
700 µL
700 µL
700 µL
Empty
Note: For best results avoid storing DNase l master mix at room temperature for long-term. It
is recommended to prepare the DNase l row A after filling Wash buffers rows D, E, F, G.
*It
is recommended to prepare the Sample row C last.
12
5. Fill the KingFisher Duo Elution strip as follows.
Elution strip
KingFisher Duo Elution strip
Content
Volume per well
Water, nuclease free
100 µL
6. Place a Thermo Scientific KingFisher Duo 12-tip comb into row B on the Plant RNA plate.
7. Switch on the KingFisher Duo instrument and ensure you are using the KingFisher Duo
12-pin magnet head and heating block. Start the Plant_RNA_Duo protocol. Insert the
Plant RNA plate and Elution strip into the instrument as indicated on the KingFisher Duo
display and press START. Make sure that the Elution strip is placed in the correct
direction into the elution block; ensure that the perforated end is facing toward the user.
8. When the KingFisher Duo pauses at the dispense step (after the DNase l digest,
approximately 30 minutes after starting the run), remove the Plant RNA plate from the
instrument and add 150 µL of Rebinding Buffer (concentrated) and 400 µL ethanol (96100%) per well to row A (DNase l row) to rebind the RNA.
Plate name and type
Plant RNA plate
Microtiter deep well 96
plate
Row
Row name
A
DNase l
Content
Volume per well
Rebinding Buffer (conc.)
150 µL
Ethanol
(96-100%)
400 µL
9. Place the Plant RNA plate back into the instrument and press OK. After the pause, the
protocol will continue through to completion.
10. After the run is complete, remove the Plant RNA plate and Elution strip according to the
instructions on the KingFisher Duo display and turn off the instrument. Transfer the
purified RNA from the Elution strip to a fresh RNase-free microtiter plate or
microcentrifuge tubes. Keep on ice for immediate use in downstream applications or store
at -70C.
13
Protocol C. Instructions for manual plant total RNA purification from up to 50 mg plant
sample
Following this protocol is based on the transfer of liquids by pipetting through different purification
steps rather than magnetic bead transfer as in the KingFisher automatic protocols. This allows the
kit to be used in various throughput applications using a magnetic rack and manual or automated
pipetting equipment. Protocols for automated pipetting platforms should be optimized for each
platform and sample type. To enable protocol optimization, all buffers are available for purchase
separately.
Note: When using the MagJET Plant RNA Kit for the first time, prepare working solutions of Wash
Buffer 1 and Wash Buffer 2 and reconstitute DNase I as described on page 5.
1. Homogenize the plant samples and prepare lysates as described on page 7-8 (SAMPLE
HOMOGENIZATION).
2. Carefully transfer 400 μL of lysate supernatant to a clean microcentrifuge tube.
3. Binding to magnetic beads: add 25 μL MagJET Magnetic beads resuspended well by
vortexing and 400 μL 96-100% ethanol. Vortex the tube and briefly spin to remove droplets from
the inside of the lid. Place the tube on the magnetic stand and let the magnetic beads collect at
the magnet for 2-3 minutes (or until the solution clears). Discard the supernatant using a pipette.
4. Wash 1: remove the tube from the magnetic stand and add 700 μL Wash Buffer 1
(supplemented with ethanol, see page 5). Resuspend the magnetic beads by vortexing, briefly
spin, then place the tube on the magnetic stand and let the magnetic beads collect at the
magnet for 2-3 minutes. Discard the supernatant using a pipette.
5. Dry: leave the tube on the magnetic rack for 5 minutes to dry the magnetic beads at room
temperature. Remove the tube from the magnetic rack.
6. DNase I treatment: add 200 µL of DNase I solution for each purification (mix 100 µL
2X DNase I Buffer, 4 µL DNase I (reconstituted), 20 µL Manganese Chloride Solution and
76 µL nuclease free water. Prepare common mix for all samples to be processed). Mix and
incubate in the thermomixer at 37°C and 350 rpm for 15 minutes.
7. Rebinding to magnetic beads: after DNase I treatment, add 150 µL of Rebinding Buffer
(concentrated) and 400 µL 96-100% ethanol. Resuspend the magnetic beads by vortexing
and briefly spin to remove droplets from the inside of the lid. Place the tube on the magnetic
stand and let the magnetic beads collect at the magnet for 2-3 minutes. Discard the
supernatant using a pipette.
8. Wash 2: remove the tube from the magnetic stand and add 700 μL Wash Buffer 1
(supplemented with ethanol, see page 5). Resuspend the magnetic beads by vortexing, place
the tube on the magnetic stand and let the magnetic beads collect at the magnet
for 2-3 minutes. Discard the supernatant using a pipette.
9. Wash 3: remove the tube from the magnetic stand and add 700 μL Wash Buffer 2
(supplemented with ethanol, see page 5). Resuspend the magnetic beads by vortexing, place
the tube on the magnetic stand and let the magnetic beads collect at the magnet
for 2-3 minutes. Discard the supernatant using a pipette.
10. Wash 4: repeat step 9 using 700 μL of Wash Buffer 2. Make sure that all the supernatant is
completely removed in the last washing step.
11. Dry: leave the tube on magnetic rack for 5 min to dry the magnetic beads at room
temperature. Remove the tube from the magnetic rack.
12. Elution: add 100 µL Nuclease free water. Resuspend the magnetic beads by vortexing,
briefly spin, then place the tube on the magnetic stand and let the magnetic beads collect at
the magnet for 2-3 minutes.
13. While on the magnetic stand, transfer the eluate (which contains the purified RNA) to a new
RNase free tube, then close immediately. Keep on ice for immediate use in downstream
applications or store at -70°C.
14
TROUBLESHOOTING
Problem
Low yield of
purified RNA
Purified RNA
is degraded
Inhibition of
downstream
enzymatic
reactions
Genomic DNA
contamination
Carryover of
the magnetic
beads in the
elution
Possible cause and solution
RNA degraded during sample storage. Make sure the plant samples were
properly stored and make sure the samples were processed immediately after
collection or removal from storage.
Insufficient homogenization of plant material. To disrupt the cell wall, it is
important to homogenize the sample thoroughly until it is ground to a fine powder.
Ethanol was not added to the lysate. Ensure that ethanol was added to the
lysate during bindind to magnetic beads step.
Incomplete resuspension of magnetic particles. Resuspend the magnetic
particles by vortexing before use.
Ethanol was not added to Wash Buffer 1: add ethanol to Wash buffer 1 as
described on page 5.
Ethanol was not added to Wash Buffer 2: ensure that ethanol was added to
Wash Buffer 2 as described on page 5.
Rebinding step was missed: ensure that rebinding buffer and ethanol was
added after DNase l treatment.
Loss of magnetic beads during purification: use sufficient magnetic beads
capture time, be careful not remove magnetic beads during procedures.
RNase contamination. To avoid RNase contamination wear gloves during the
procedure and change gloves frequently. Use sterile, disposable RNase-free
pipette tips. Use reagents designed to remove RNase contamination from
nondisposable items (pipettes, centrifuges) and work surfaces.
RNA degraded during sample storage. Make sure the plant samples were
properly stored and make sure the samples were processed immediately after
collection or removal from storage.
RNA degraded during sample homogenization. To minimize RNA degradation,
avoid repeated freezing and thawing of the samples and perform extractions from
fresh material or material that has been immediately frozen and stored at -70°C.
Transfer the ground tissue to the Lysis Buffer for MagJET Plant RNA Kit as quickly
as possible to avoid RNA degradation. All ground material must be thoroughly
mixed with the Lysis Buffer for MagJET Plant RNA. RNA degradation can occur in
particles that are left to dry on the walls of the tube.
Lysis Buffer was not supplemented with DTT. Ensure that 2 M DTT solution
has been added to the Lysis Buffer before RNA purification procedure.
Purified RNA was not stored properly. Purified RNA should be used immediately in
downstream applications or stored at -20°C for later use. For prolonged storage
(more than 1 month) storage at -70°C is recommended.
Purified RNA contains residual salt. Use the correct order of Wash Buffers
steps. Always wash the magnetic beads with Wash Buffer 1 first and then proceed
with Wash Buffer 2.
Purified RNA contains residual ethanol. It is important to dry magnetic beads
before elution step.
Inefficient DNase I digestion. Ensure that the temperature of DNase I digestion is
37°C. Prepare DNase I mix according to protocol (see p. 7 and p. 10).
Too much sample input: if starting amount of plant sample is too high (> 50 mg),
the DNA digestion step may not be effective.
Carryover of magnetic beads in the eluted RNA will not affect downstream
applications. To remove the carryover from eluted RNA, simply magnetize the
magnetic beads and carefully transfer eluate to a new tube or plate.
15
SAFETY INFORMATION
Lysis Buffer for MagJET Plant RNA Kit
Xn Harmful
Hazard-determining components of labelling: guanidinium thiocyanate
Risk phrases:
R20/21/22: Harmful by inhalation, in contact with skin and if swallowed.
R32-52/53: Contact with acids liberates very toxic gas. Harmful to aquatic organisms, may cause long-term
adverse effects in the aquatic environment.
Precautionary statements
P261 Avoid breathing dust/fume/gas/mist/vapours/spray.
P280 Wear protective gloves/protective clothing/eye protection/face protection.
P305+P351+P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if
present and easy to do. Continue rinsing.
P301+P312 IF SWALLOWED: Call a POISON CENTER or doctor/physician if you feel unwell.
P304+P340 IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing.
P501 Dispose of contents/container in accordance with local/regional/national/international regulations.
Wash Buffer 1 (conc.) for MagJET Plant RNA Kit
Xn Harmful
Hazard-determining components of labelling: guanidinium chloride
Risk phrases:
22 Harmful if swallowed.
36/38 Irritating to eyes and skin.
Safety phrases:
23 Do not breathe gas/fumes/vapour/spray.
26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
36/37 Wear suitable protective clothing and gloves.
60 This material and its container must be disposed of as hazardous waste
Rebinding Buffer for MagJET Plant RNA Kit
Xn Harmful
Hazard-determining components of labelling: guanidinium chloride
Risk phrases:
22 Harmful if swallowed.
36/38 Irritating to eyes and skin.
Safety phrases:
23 Do not breathe gas/fumes/vapour/spray.
26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
36/37 Wear suitable protective clothing and gloves.
60 This material and its container must be disposed of as hazardous waste
PRODUCT USE LIMITATION
This product is developed, designed and sold exclusively for research purposes and in vitro use only. The
product was not tested for use in diagnostics or for drug development, nor is it suitable for administration to
humans or animals.
Please refer to www.thermoscientific.com/onebio for Material Safety Data Sheet of the product.
© 2013 Thermo Fisher Scientific Inc. All rights reserved. All (other) trademarks are the property of Thermo
Fisher Scientific Inc. and its subsidiaries.
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